Direct write nanolithographic deposition of nucleic acids from nanoscopic tips

ABSTRACT

The use of direct-write nanolithography to generate anchored, nanoscale patterns of nucleic acid on different substrates is described, including electrically conductive and insulating substrates. Modification of nucleic acid, including oligonucleotides, with reactive groups such as thiol groups provides for patterning with use of appropriate scanning probe microscopic tips under appropriate conditions. The reactive groups provide for chemisorption or covalent bonding to the substrate surface. The resulting nucleic acid features, which exhibit good stability, can be hybridized with complementary nucleic acids and probed accordingly with use of, for example, nanoparticles functionalized with nucleic acids. Patterning can be controlled by selection of tip treatment, relative humidity, and nucleic acid structure.

This application is a divisional of application Ser. No. 10/307,515,filed Dec. 2, 2002 and claims benefit of provisional application Ser.No. 60/337,598, filed Nov. 30, 2001, (“Patterning of Nucleic Acids byDip-Pen Nanolithography” to Mirkin et al.), and of provisionalapplication Ser. No. 60/362,924 filed Mar. 7, 2002, (“Direct Patterningof Oligonucleotides on Metal and Insulator Surfaces via Dip-penNanolithography”), the complete disclosure of which is herebyincorporated by reference.

This invention was made with government support under Grant Nos:EEC011802 from the National Science Foundation, F49620-00-1-0283 fromthe Air Force Office of Scientific Research, HG02463 from the NationalInstitute of Health, and DAAG55-97-01-0133 from the Army ResearchOffice. The government has certain rights in the invention.

BACKGROUND

Nanotechnology has many applications throughout biology, biochemistry,chemistry, medicine, genetics, diagnostics, and therapeutics. Inaddition to generating totally new technologies, nanotechnology alsopromises to further enable existing technologies to be miniaturized tosub-micron levels. Too often, technology is limited to the micron scale.For example, nucleic acid microarrays have been commercialized at amicron level for biological and genetics applications includingdocumenting gene expression on a genome-wide scale (see, for example, APrimer of Genome Science, G. Gibson and S. Muse, 2002, Chapters 3-4).Present microarrays include the cDNA and oligonucleotide types. A strongcommercial need now exists, however, to make arrays on a much smaller,nanometer scale, particularly at lateral dimensions of less than about100 nm. In other words, nucleic acid nanoarrays are needed with muchhigher densities of sample sites, which approach the size of singlemolecules, monolayers, and sub-100 nm dimensions. Production methodsused to produce microarrays, however, generally are not capable ofnanoarray production. Moreover, currently used robotic printing of thesearrays can suffer from the printing pins being expensive, fragile, proneto clogging, poor uniformity, and tendency to deliver doughnut-shapedspots as the nucleic acid spreads away from the tip. In addition tonucleic acid arrays, peptide arrays are also important, and at times,combined nucleic acid and peptide structures are of interest. Hence,novel nanotechnology is needed which enables the production ofnanoarrays at a commercial level and pushes the limits of microarrayminiaturization. In particular, difficulties become more severe whenbreaking the sub-100 nm barrier, when entering the realm of singlemolecules and monolayers, and when entering the commercial marketplace.

Nanoscopic tips including scanning probe microscopic (SPM) tips havegenerally been used to characterize nanoscale structures but their usein fabrication at the nanoscale is much less developed. Early attemptsat fabrication were not successful. A need exists to make better use ofnanoscopic SPM tips in nanoscale fabrication including, for example, theproduction of nanoarrays with applications both in the biological andnon-biological arts. SPM tips are of particular interest if they can beused for direct writing and patterning of substrates at a molecularlevel. The challenge of direct writing at this level is particularlysignificant for direct writing of biological compounds including nucleicacids. Improvements are needed which provide, for example, betterresolution, higher reproducibility, better stability, and betterretention of molecular recognition and hybridization. One particularlyimportant challenge is the direct writing of single nucleic acidstrands, wherein molecular size and charge effects may become important,factors which generally are less relevant for direct writing ofuncharged, small molecules. Indirect methods are known for generation ofnucleic acid structures at small scales, wherein for example nucleicacids are absorbed to existing lithographic features. Nevertheless,direct writing provides significant advantages over indirect pathways.

One method for direct write nanolithography is DIP PEN NANOLITHOGRAPHY™printing and deposition (i.e., DPN™ printing and deposition), which isdescribed further below and is being developed at NorthwesternUniversity in the Mirkin group and at NanoInk, Inc. (Chicago, Ill.).DPN™ and DIP PEN NANOLITHOGRAPHY™ are trademarks of NanoInk, Inc. Thismethod is versatile and can be carried out with readily accessibleequipment. Complicated stamps and resists are not generally needed.Despite the success of this technology to date, improvements are stillneeded.

Finally, interest in generating nucleic acid features on a nanoscalealso arises because of the ability these compounds have to recognize andbind to complementary strands of nucleic acid (i.e., hybridize) whichcould provide for “bottom-up” nanoscale manufacturing of functionalmaterials including molecular electronic and photonic devices.Programmed materials synthesis with DNA is described in, for example,Mirkin, Inorganic Chem., 2000, 39, 2258-2272; Mirkin, MRS Bulletin,January, 2000, pgs. 43-54; and Storhoff et al., Chem. Rev., 1999, 99,1849-1862. Also, hybridization of nucleic acids is discussed in thecontext of surface-confined DNA probe arrays in, for example, Heme etal., J. Am. Chem. Soc., 1997, 119, 8916-8920; Levicky et al., J. Am.Chem. Soc., 1998, 120, 9787-9792. Diagnostic applications are alsoimportant as discussed in, for example, U.S. Pat. No. 6,361,944 toMirkin et al. (Nanosphere, Inc.).

SUMMARY

In this section, the inventions disclosed herein are summarized, butthis summary does not limit the scope of the invention, which isdescribed in detail and claimed further below.

The present invention, briefly, provides for a method for depositingnucleic acid onto a substrate by direct-write nanolithography comprisingthe step of positioning at least one nanoscopic tip relative to asubstrate so that the tip approaches the substrate, wherein nucleic acidis transferred from the tip to the substrate to generate a stablenucleic acid nanoscale pattern which can be hybridized withcomplementary nucleic acid.

Briefly, the present invention also provides a method for generatingnanoscale patterns of nucleic acid on a substrate comprising positioninga scanning probe microscopic tip relative to the substrate so that thetip approaches the substrate at a relative humidity sufficiently high sothat nucleic acid is transferred from the tip to the substrate to form ananoscale pattern, wherein before transfer the tip is modified to allowthe nucleic acid to wet the tip and the nucleic acid is modified tochemisorb or covalently bond to the substrate.

Briefly, the present invention also provides a method for directpatterning of modified nucleic acid onto a substrate comprising thesteps of inking a scanning probe microscopic tip with a modified nucleicacid and positioning the inked tip close enough to the substrate toeffect transfer of the nucleic acid to the substrate to form a nanoscalepattern, wherein the nucleic acid is modified with a functional groupwhich provides for chemisorption or covalent bonding to the substrate,and the functional group is bonded to the nucleic acid via a spacer.

Still further, the invention also provides a method for directpatterning of modified nucleic acid onto a substrate comprising the stepof positioning a scanning probe microscopic tip inked with a modifiednucleic acid and positioning the inked tip close enough to the substrateto effect transfer of the nucleic acid to the substrate to form ananoscale pattern, wherein the nucleic acid is modified with anelectrophilic or nucleophilic functional group which provides forcovalent bonding to the substrate.

In addition, the invention, briefly, provides a method for improving thetransfer of nucleic acid from a scanning probe microscopic tip to asubstrate during direct write nanolithography comprising modifying thetip to make it positively charged.

Briefly, the invention also provides a method for improving direct writedeposition of nucleic acid from a scanning probe microscope tip to asubstrate comprising the step of treating the tip with one or morecompositions which improves adhesion of the nucleic acid to the tip.

The invention also provides, briefly, a method for assemblingnanoparticles to form nanoscale patterns comprising the steps of: (a)depositing from a nanoscopic tip a first nucleic acid onto a substrateto form a deposit with lateral nanoscale features of about 1,000 nm orless by direct write nanolithography; (b) hybridizing the nucleic aciddeposit with the nanoparticle, wherein the nanoparticle isfunctionalized with a second nucleic acid which is either (1)complementary to the first, or (2) complementary to the nucleic acid ofa linking strand which links the second nucleic acid to the first.

Further, the invention also provides a method for assemblingnanoparticles to form nanoscale patterns comprising the step ofhybridizing a nucleic acid nanoscale deposit on a substrate, the depositcomprising a first nucleic acid, with a nanoparticle, wherein thenanoparticle is functionalized with a second nucleic acid which iseither (1) complementary to the first, or (2) complementary to thenucleic acid of a linking strand which links the second nucleic acid tothe first.

Still further, the invention provides a nanoscale nucleic acid patternon a substrate comprising the substrate and at least one pattern of afirst nucleic acid on the substrate, wherein the pattern of firstnucleic acid is chemisorbed or covalently bonded to the substrate, has alateral dimension of 1,000 nm or less, and is hybridizable to a secondnucleic acid complementary to the first.

The invention provides a nucleic acid nanoarray comprising a substrateand a plurality of patterns of nucleic acid on the substrate, whereinthe patterns of nucleic acid are chemisorbed or covalently bonded to thesubstrate, have lateral dimensions of about 1,000 nm or less and areseparated from each other by distances of 1,000 nm or less, have apattern density of at least 100,000 per square centimeter, and arehybridizable to complementary nucleic acids.

In addition, the invention also includes articles comprising substrateswith nucleic acid patterns thereon, nucleic acid nanoarrays, scanningprobe microscopic (SPM) tips coated with nucleic acid, solutions used tocoat SPM tips, kits for direct write nanolithography of nucleic acids,and computer software for same.

Basic and novel features of the invention, which are discussed in detailbelow, are many and include the advantages of DPN printing alreadyestablished in the art including the ability to directly writepreconceived nanoscale features without use of expensive and potentiallydestructive methods such as electron beam and photolithographic methods.Also, the structures can be built up, if desired, without degradingexisting structures. Complicated stamps and resists are not needed.Improvements in the consistency and stability of the nanolithography canbe observed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates coating a silicon nitride tip with a hexanethiol andpolyethylene glycol terminated single stranded DNA.

FIG. 2 illustrates use of DNA complementary linker strands to directparticle assembly onto nanoscale features. Particle assembly does notoccur with non-complementary linker strand or with no linker strand.

FIG. 3 illustrates transport of cyclic disulfide-modified DNA to a goldsubstrate from a coated AFM tip.

FIG. 4 illustrates lateral force microscopy images of DNA patterns.Control over DNA pattern size was achieved by varying relative humidityin an atmospheric control chamber

FIG. 5 illustrates a tapping mode AFM image of DNA patterns generated ongold via DPN (lines) and 13 nm gold nanoparticles assembled onto thepatterns via specific DNA hybridization interactions.

FIG. 6 illustrates direct transfer of DNA onto gold substrates by DPNprinting. (A) Tapping-mode AFM image of hexane-thiol-modifiedoligonucleotides patterned on polycrystalline gold. The scale barrepresents 2 microns, and the space between the arrows is 150 nm. (B)Tapping-mode AFM images of single oligonucleotide-modified goldnanoparticles (13 nm diameter) bound to a high resolution DNA line ongold by Watson-Crick base pairing in the presence of complementarylinking DNA. The scale bar represents one micron.

FIG. 7 illustrates tapping-mode AFM phase image of hexanethiol-modifiedoligonucleotides patterned on polycrystalline gold after exposure tooligonucleotide-modified gold nanoparticles (13 nm diameter)prehybridized to a 24-base linker sequence which was not complementaryto the patterned DNA.

FIG. 8 illustrates direct transfer of hexanethiol-modifiedoligonucleotides onto polycrystalline gold substrates via DPN printing.(A) Lateral force atomic force micrograph of patterned surface after ODTtreatment, (B) tapping-mode AFM image of a DNA pattern after ODTtreatment.

FIG. 9 illustrates tapping-mode AFM image of (A) single G1-modified goldnanoparticles (13 nm diameter) bound to DNA lines (50 nm wide) of C1 ongold via Watson-Crick base pairing in the presence of complementarylinking DNA, L1 (B) G1-modified gold nanoparticles bound to thick DNAlines via hybridization in the presence of L1.

FIG. 10 illustrates direct DPN transfer of DNA onto insulatingsubstrates. (A) Epifluorescence micrograph of fluorophore-labeled DNA(Oregon Green 488-X) hybridized to a DPN-generated pattern ofcomplementary oligonucleotides on an SiO_(x) surface. The scale barrepresents 12 microns. (B) Tapping-mode AFM image ofoligonucleotide-modified gold nanoparticles (13 nm diameter) hybridizedto a second, high resolution pattern after removal (using DI water) ofthe fluorophore-labeled DNA. The scale bar represents 1.5 microns, andthe space between the arrows is 100 nm.

FIG. 11 illustrates a scheme for the DPN printing functionalization ofSiOx substrates. (A) Silicon wafers are treated with3-mercaptopropyltrimethoxysilane, (B) a DNA (SEQ ID No: 7) coated AFMtip delivers acrylamide modified DNA to the substrate during a briefcontact (seconds).

FIG. 12 illustrates DPN printing control over deposited DNA featuresize. (A) Tapping-mode AFM image of thiol-modified DNA deposited on agold substrate for different tip contact times at 45% relative humidity.(B) Spot diameter (from A and duplicate experiments) plotted vs. squareroot of contact time. Error bars were calculated from the standarddeviation of at least 5 points.

FIG. 13 further illustrates DPN printing control over deposited featuresize. (A) Tapping-mode AFM image of thiol-modified DNA spotted on a goldsubstrate for different contact times at 45% relative humidity (top) andplot of dot diameter versus square root of contact time (bottom). (B)Tapping-mode AFM image of nanoparticles hybridized to DNA spots formedon SiO_(x) for different contact times at 45% relative humidity (top)and plot of dot diameter versus square root of contact time (bottom).The scale bars for (A) and (B) represent 2 microns. (C) Tapping-mode AFMimage of DNA spots generated on polycrystalline Au with contact time of10 s per spot, at varying relative humidity (top), and a plot of spotdiameter versus relative humidity (bottom). The scale bar represents 1micron. Error bars for all plots were calculated from the standarddeviation of at least five points.

FIG. 14 illustrates the humidity dependence of DNA transport rate.Tapping mode AFM images of (A) DNA spots generated on polycrystalline Auwith contact time of 10 s/spot, from 30-46% relative humidity (RH)(bottom to top); (B) 13 nm gold particles hybridized to DNA spotsgenerated with contact time of 10 s/spot, from 50-80% relative humidity(bottom to top); (C) spot diameter vs. relative humidity for points in Aand B.

FIG. 15 illustrates direct patterning of multiple-DNA inks by DPNprinting. (A) Combined red-green epifluorescence image of two differentfluorophore-labeled sequences (Oregon Green 488-X and Texas Red-X)simulataneously hybridized to a two-sequence array deposited on an SiOxsubstrate by DPN printing. (B) Tapping mode AFM image of 5 (dark)- and13 (light)-nm diameter gold nanoparticles assembled on the same patternafter dehybridization of the fluorophore-labeled DNA. The scale barrepresents 4 microns. (C) The line plot was taken diagonally throughboth nanoparticle patterns, and the start and finish are indicated bythe arrows in (B). The scale bar represents 4 microns.

FIG. 16 illustrates AFM images of patterned gold substrates. (A)Tapping-mode AFM image of DNA patterns, C1 (square dot array) and C2(triangular dot array) after ODT passivation. (B) Tapping-mode AFM imageof 13 nm and 30 nm diameter gold nanoparticles hybridized selectively toC1 and C2 DNA patterns in the presence of L1 and L2 respectively. (C)Line scan showing height profile across patterns in A afterhybridization to 13 and 30 nm diameter particles.

DETAILED DESCRIPTION

DPN printing, patterning, and deposition methods are disclosed in, forexample, the following references from the Mirkin group, which arehereby incorporated by reference in their entirety: (1) Piner et al.,Science, 283, Jan. 29, 1999, page 661; (2) Hong et al., Science, 286,Oct. 15, 1999, page 523; and (3) Hong et al., Science, 288, Jun. 9,2000, page 1808. Further, the technical publication “Direct Patterningof Modified Oligonucleotides on Metals and Insulators by Dip-PenNanolithography”, by Demers et al., Science, Vol. 296, Jun. 7, 2002,pgs. 1836-1838, is also hereby incorporated by reference in itsentirety, including the supporting online material cited therein. DPNprinting and patterning of nucleic acids, as well as nanoparticulatenucleic acid probes, have been noted in the following references, whichare hereby incorporated by reference: (1) C. A. Mirkin, Mater. Res. Soc.Bull., 25, 43 (2000), (2) C. A. Mirkin, Inorg. Chem., 39, 2258 (2000).

Also, provisional patent application Ser. No. 60/337,598 to Mirkin etal, filed Nov. 30, 2001 entitled “Patterning of Nucleic Acid by Dip PenNanolithography” is hereby incorporated by reference in its entirety.

Also incorporated by reference in its entirety is the Ph.D. thesis by L.M. Demers, Northwestern University, June 2002, “Nanolithography andBiomolecular Recognition as Tools for the Directed Assembly and Study ofParticle-Based Materials,” Chapter 6, “Direct-Patterning of DNA viaDip-Pen Nanolithography.”

Materials, devices, instruments, software and hardware, articles, andconsultation related to DPN printing are also available from NanoInk,Inc. (Chicago, Ill.).

Additional technical publications relating to SPM probes and nucleicacid deposition and which are hereby incorporated by reference include:(1) “Meniscus Force Nanografting: Nanoscopic Patterning of DNA,”Schwartz, Langmuir, 2001, 17, 5971-5977; (2) “Molecular Transport froman Atomic Force Microscope Tip: A Comparative Study of Dip-PenNanolithography,” Schwartz, Langmuir, 2002, 18, 4041-4046; and (3) WO02/45215 A2 with international PCT publication date of Jun. 6, 2002 toMirkin, Schwartz, et al. “Nanolithography Methods and Products Thereforand Produced Thereby.” The latter PCT publication, for example,discloses use of patterning solutions comprising nucleic acid and salt,including cationic surfactants and ammonium compounds such as, forexample, tridodecylmethylamine. The solutions can be aqueous and can beused to coat the SPM tip.

The role of biomolecules in materials applications is disclosed in thefollowing references, which are hereby incorporated by reference: (1) J.J. Storhoff, C. A. Mirkin, Chem. Rev., 99, 1849 (1999); (2) C. M.Niemeyer, Angew. Chem. Int. Ed. 40, 4128 (2001).

In U.S. patent application Ser. No. 09/866,533, filed May 24, 2001 (seealso corresponding U.S. patent publication US 2002/0063212 A1 to Mirkinet al. published May 30, 2002), DIP PEN™ nanolithographic printingbackground and procedures are described in detail covering a widevariety of embodiments including, for example:

-   -   background (pages 1-3);    -   summary (pages 3-4);    -   brief description of drawings (pages 4-10);    -   use of nanoscopic scanning probe microscope tips (pages 10-12);    -   substrates (pages 12-13);    -   patterning compounds including oligonucleotide, DNA, and RNA        (pgs. 13-17);    -   practicing methods including, for example, coating tips (pages        18-20);    -   instrumentation including nanoplotters (pages 20-24);    -   multiple layers and related printing and lithographic methods        (pages 24-26);    -   resolution (pages 26-27);    -   arrays and combinatorial arrays (pages 27-30);    -   software and calibration (pages 30-35; 68-70);    -   kits and other articles, tips coated with hydrophobic compounds        (pages 35-37);    -   seven working examples (pages 38-67);    -   corresponding claims and abstract (pages 71-82); and    -   FIGS. 1-28.

All of the above text, including each of the various subsectionsenumerated above including the figures, is hereby incorporated byreference in its entirety and form part of the present disclosure,supporting the claims.

In addition, U.S. Patent Publication 20020122873 A1 (application serialno. 059,593 filed Jan. 28, 2002) to Mirkin et al (published Sep. 5,2002) is also incorporated by reference in its entirety. It discloses,for example, use of driving forces to control the movement of adeposition or patterning compound from a nanoscopic scanning probemicroscopic tip to a substrate. It also discloses a tip having aninternal cavity and an aperture restricting movement of a deposition orpatterning compound from the tip to the substrate. The rate and extentof movement of the deposition or patterning compound through theaperture can be controlled by the driving force. Nucleic acid can bedeposited or patterned by this method using, for example, a positivelycharged substrate which attracts the negatively charged nucleic acid.Aperature Pen Nanolithographic methods and fountain pen nanolithographicmethods can be used to deposit or pattern nucleic acid as describedherein.

DIP PEN nanolithographic printing, and the aforementioned procedures,instrumentation, and working examples, can be adapted also to generateimproved nucleic acid and DNA articles and nanoarrays as describedfurther herein. As described further herein, the nucleic acid can bevaried widely, but oligonucleotides are of particular interest and, inparticular, oligonucleotides which have been modified or have chemicalstructures which provide for covalent bonding or chemisorption to thesubstrate surface.

To transfer the nucleic acid from the nanoscopic tip to the substrate asa pattern or deposit, the substrate and tip are moved in relation toeach other so that they approach each other. The tip can be moved towardthe substrate, the substrate can be moved toward the tip, or both thetip and substrate can be moved toward each other.

In general, nanoscopic, submicroscopic tips can be used which arecapable of delivering, patterning, or depositing nanoscopic,submicroscopic amounts of nucleic acid patterning compound from the tipto the substrate. Although the nanoscopic tip design is not particularlylimited, in general, the nanoscopic tip can have a tapered orsubstantially tapered end point which is characterized by nanometer,sub-micron level dimensions rather than microscopic dimensions. Forexample, there can be nanoscopic level tip widths, cavity, or aperturediameters. In general, nanoscopic tips are preferred which are capableof both imaging nanoscopic structures and depositing nanoscopicstructures. SPM tips can be used, and a preferred type of SPM tip is theatomic force microscope (AFM) tip. In general, tips can be used whichare designed so that they can be coated with ink on their outer surfacesuch as, for example, atomic force microscope tips. Alternatively, tipscan be hollow, including tips which have an aperture opening or NSOMtips. The ink can be delivered continuously if desired. An array of tipscan be used, as known in the art, wherein each tip can be individuallycontrolled or collectively operated as desired. The tip can be attachedto a cantilever or a functional equivalent thereof.

Known SPM and AFM methods can be used including, for example, contact,non-contact, tapping, and lateral force modes.

In studying direct transfer of nucleic acid from an SPM tip to asubstrate, several factors can be important which promote nucleic acidpatterning into consistent, high quality patterning. These factors arealso discussed further in the Working Examples section.

First, for example, the tip can be well-coated with nucleic acid. Forexample, surface-modification of a conventional silicon nitride AFMcantilever can promote reliable adhesion of the nucleic acid ink to thetip surface. See, for example, FIG. 1. The tip can be modified at thetip surface to have a positive charge including, for example, a positivecharge of amino or ammonium groups. Single or multi-step processes canbe used to generate tips designed for nucleic acid deposition. Forexample, a pretreatment step can be carried out which allows the tip tobe functionalized in a subsequent step. For example, the tip can bemodified to provide a hydroxylated surface and then the hydroxy groupscan be further functionalized. The tip can be treated with strong acidand peroxide, including for example sulfuric acid and hydrogen peroxide.After pretreatment, tip functionalization can be accomplished bytreating the tips with, for example, a silane coupling agent such as anaminosilane coupling agent, 3′-aminopropyltrimethoxysilane (for example,1-2 h, 1% v/v solution in toluene).

The silanized tips can then be coated with nucleic acid. Coating can becarried out with use of a solution comprising nucleic acid and salt. Thesolvent can be an aprotic solvent such as DMF which can be further mixedwith water as desired. In general, more aprotic solvent can be used thanwater so that, for example, about 70% to about 90% by weight of solventcan be aprotic. The salt can be an inorganic salt such as a group IIsalt or a halide salt including, for example, magnesium chloride. Theconcentration of the salt can be, for example, about 0.01 M to about 0.4M, more particularly about 0.1 M to about 0.2 M. The ratio of the saltto the nucleic acid can be varied to provide good deposition conditionsand good nanoscopic structures. The concentration of the nucleic acidcan be, for example, about 0.1 mM to about 10 mM, and more particularly,about 0.5 mM to about 5 mM.

The tips can be treated by dipping them for less than a minute asdesired, for example about 20 s, into the nucleic acid tip coatingsolution (see Working Examples), and then blowing dry briefly withcompressed gas such as, for example, difluoroethane. AFM tips preparedand coated with nucleic acid in this way can be used in direct writeprinting experiments for several hours, as described herein, before theycan be recoated. In addition, tips can be recoated and used again forthe same nucleic acid sequence.

Second, control of ambient relative humidity can provide consistent,high quality direct write patterning of nucleic acid. The relativehumidity can be controlled to be sufficiently high to provide transferof the nucleic acid from the tip to the substrate. In general, relativehumidities of at least about 25% can be used, and more particularly,about 25% to about 100%, and more particularly, about 40% to about 100%,and more particularly, about 40% to about 50%. For example, ambientpatterning can be performed in an environmentally controlled glovebox ata relative humidity of 45±5% at 23±3° C. Relative humidity can becontrolled based on other deposition factors which include the nature ofthe nucleic acid, the nature of the substrate, the nature of the desiredpattern (e.g., dot or line), the nature of the nucleic acid solutionused to wet the tip, the nature of the tip, and the like. The role of ameniscus can be important with direct write nanolithographic printingusing SPM tips, and the size of the meniscus can be related to therelative humidity.

In addition to tip-coating and humidity, selection of the ink-substratecombination can also facilitate the direct writing of nucleic acid intoconsistent, high quality features. For example, the nucleic acid can bemodified to include functional groups which chemisorb or covalently bondto the substrate. A variety of approaches can be used. For example, thefunctional group for binding the substrate can be directly bonded to thenucleic acid or can be linked to the nucleic acid by a relativelyflexible spacer group. Examples of spacer groups include flexible chainoligomers such as alkylene glycols, for example, polyethylene orpolypropylene glycol. These can have, for example, 3-20 alkyleneoxyrepeat units. The functional group can be, for example, asulfur-containing moiety such as thiol or disulfide designed tochemisorb to gold surfaces. The procedures can be used to pattern cyclicdisulfide-modified nucleic acid and trithiol-modified nucleic acid. Thesulfur atom can be bound to hydrocarbon groups such as, for example,alkyl groups, including C₄-C₁₈ alkyl groups. The functional group canbe, for example, an electrophilic group designed to react with anucleophilic surface, or a nucleophilic group designed to react withelectrophilic surfaces. Michael addition reaction, for example, can beused to bond the nucleic acid to the substrate.

For example, hexanethiol, PEG-modified oligonucleotides can be used todirectly pattern gold substrates with features ranging from about 50 nmto several micrometers in size. The hexanethiol group of the nucleicacid can provide for chemisorption to the underlying Au surface. Otherembodiments which allow the nucleic acid to be modified with functionalgroups for attaching to the substrate include (1) phosphorothioategroups (see, e.g., U.S. Pat. No. 5,472,881), (2) substituted siloxanesincluding aminosiloxanes and mercaptoalkylsiloxanes, (3) embodimentsdescribed in U.S. Pat. No. 6,361,944, which is hereby incorporated byreference. Types of nucleic acids and their possible modifications arealso described further below.

The substrates can be treated so they comprise both nucleic acid and apassivation agent on the substrate. For example, after the substratesare patterned with nucleic acid, they can be passivated. In onepassivation embodiment, unpatterned areas of the substrate can betreated with a passivation agent so as to reduce the reactivity of theunpatterned areas of the substrate during further processing.Passivation can be carried out for a number of reasons including, forexample, improving the analysis of or detection of the patterned nucleicacid. For example, if hybridization of the nucleic acid is desired bycomplementary nucleic acid, then the selectivity of the interactionbetween the nucleic acid patterned areas and the unpatterned areas canbe improved through passivation. Passivation can be carried out byimmersing the patterned substrates in solutions wherein the solutioncontains a passivation agent such as an alkane thiol which selectivelyadsorbs to the unpatterned area of the substrate such as gold. Hence,the passivation agent can comprise one reactive functional group whichprovides for chemisorption or covalent bonding to the unpatternedsubstrate, but does not have other functional groups. For example, thepassivation agent can comprise a long chain alkyl group which uponadsorption exposes methyl groups to the surface which are generallyunreactive to subsequent processing such as nucleic acid hybridization.The passivation can make the rest of the substrate hydrophobic forexample. For example, a gold substrate which has already been patternedwith a nucleic acid can be immersed in an ethanol solution of1-octadecanethiol (ODT, 1 mM) for 1 min. This procedure coats theunpatterned gold surface with a hydrophobic monolayer, passivating ittowards the non-specific adsorption of DNA or DNA-modified nanoparticlesin subsequent hybridization experiments.

In general, the passivation agent can displace the patterned nucleicacid if the nucleic acid is not functionalized to chemisorb to orcovalently bond to the substrate. For example, nucleic acid which doesnot contain a thiol moiety can be displaced from the surface during theODT treatment.

In another passivation embodiment, the substrate is first patterned withthe passivation agent, followed by patterning with nucleic acid. Inother words, substrates can be passivated before patterning. Forexample, substrates can be treated with a passivation agent such as, forexample, an adsorption resistant hydrogel to which oligonucleotides andother nucleic acids can be bound. Passivation agents known in the art ofmicroarray technology can be used.

The nucleic acid patterns can be imaged by tapping mode AFM, forexample, following the passivation of the substrate by, for example, ODTtreatment. This imaging can provide height measurements. In general, thefeature height of the nucleic acid pattern as measured by AFM or similartechnique can be, for example, about 100 nm or less, or moreparticularly, about 10 nm or less. For oligonucleotides, height can be,for example, about 2 nm to about 5 nm.

To demonstrate that the patterned, immobilized nucleic acid can retainits highly-specific recognition properties and is accessible tohybridization, nucleic acid patterns can be used to direct the assemblyof nanoparticles which comprise complementary nucleic acid (see forexample FIG. 2). Structures can be fabricated via this process on themicron to sub-100 nm length scale. The high resolution thus affordedallows control over the placement of individual particles on a surfacein the form of a preconceived architecture. Also, the nucleic aciddensity on the patterns can be sufficiently high to bind nanoparticlesin a closely-packed arrangement.

In particle assembly, a three-component system can be applied: (1)patterned nucleic acid, (2) linker nucleic acid, and (3) particle-boundnucleic acid, as described in FIG. 2 and further described in theWorking Examples (see also abbreviations therein for C, G, and L typesof nucleic acid used below). In this approach, there is a built-incontrol experiment where the linker can be left out or replaced with anon-complementary sequence. In one such control experiment, for example,15-mer DNA (G1) modified gold nanoparticles can be hybridized to a30-mer oligonucleotide, L2, the first 15 bases of which werecomplementary to the particle-bound DNA. However, the free 15-basesegment of the linker can be made not complementary (less than 4consecutive base overlap) to the DNA on the patterned surface, C1.

The DNA-patterned surfaces can be exposed to non-complementaryparticle-linker solution under hybridization conditions (e.g., 0.3 MPBS, 0.025% SDS, 3 h, room temperature), but particles bind to thepattern in at most only a few places. Hybridization conditions are knownin the art and are described in, for example, U.S. Pat. No. 6,361,944,which is hereby incorporated by reference. A phase AFM image rather thana topography image can be used because better contrast between ODTmonolayer, DNA pattern, and gold nanoparticles can be achieved usingthis imaging mode. Thus, even in the absence of stringency (highertemperature) washes the interactions between the DNA nanopatterns andthe oligonucleotide-modified nanoparticles are highly selective,precluding adsorption of particles that are modified withnon-complementary DNA. In contrast, a high particle density can beobserved on samples where the correct complementary linkeroligonucleotide is present.

The stability of patterned nucleic nanostructures on the substrate canbe tested by using a patterned surface for particle assembly after it isstored under ambient conditions for more than 3 months. Significantly, ahigh degree of specific particle adsorption indicates that the patterncan be for the most part intact, with some patchy areas which may be dueto degradation of either the nucleic acid, or loss of thiol-modifiednucleic acid from the surface. In addition, there can be a smallincrease in the number of nanoparticles bound to the ODT monolayerregions of the chip (fresh samples exhibit nearly perfectlyparticle-free backgrounds). A substantial decrease in particlebackground can be achieved if samples are stored in an inert atmospherein the dark in order to minimize air and/or photooxidation of thethiol-Au linkages.

An important feature of DPN printing is the ability to generate patternsof specific chemical functionality over a large range of length scales(sub-100 nm to many micron) while exhibiting fine control over featuresize. Using the methods described herein, highly-charged macromoleculessuch as nucleic acid and oligonucleotides can be transferred to asubstrate from an SPM tip in much the same way as small hydrophobicmolecules.

Specifically, using the methods described herein, the pattern spot sizecan increase as a function of tip-surface contact time or,alternatively, the line width can be increased by slower draw speeds.For example, the relative humidity can be kept constant at, for example,45% and nucleic acid spots can be formed by holding a C1 DNA-coated AFMtip at different points on an Au substrate for fixed contact times(minimum of 5 spots at each contact time, ranging from 0.1 s to 100 s).Under these conditions, the transport of DNA from the AFM tip to thesurface can follow the same linear increase in pattern area with contacttime (spot diameter˜t^(1/2)) predicted by theoretical simulations aswell as experimentally observed for smaller molecules. Although the rateconstants can be different for each ink-substrate pair, this underscoresthe control DPN printing can offer for patterning compounds ranging fromsmall molecules and salts to organic, charged macromolecules on avariety of substrates.

Also, the rate of nucleic acid pattern formation on gold substrates canbe tailored with careful humidity control using the methods describedherein. To illustrate and quantify this effect, a series of dots can beformed by holding the tip in contact with a gold substrate for, forexample, about 10 s while varying the relative humidity (RH) in theglove box for each spot. Humidity can be increased by, for example,bubbling nitrogen through a container of water and flowing the vaporinto the box. The humidity can be kept stable by an automatic controllerwhich alternatively flowed dry nitrogen or water-saturated nitrogenthrough the box. Before patterns are made, the humidity can be allowedto equilibrate for, for example, at least 5 min at each point afterhygrometers placed at the ceiling and floor of the box read the samevalue (±0.5%). In two separate embodiments (different substrates on adifferent days, but using the same AFM tip and DNA sequence, (C1) RH canbe changed from about 30% to about 46%. Hence, feature size can bevaried over a large dynamic range on a reasonable timescale usinghumidity control. For example, the diameter of a spot created by holdingthe AFM tip for 10 s can change from less than 50 nm to 1000 nm with aRH increase of 50%. In addition, the affect of relative humidity on thepattern spot size for a given contact time can be well defined. Thepattern area can vary roughly as the square of the relative humidity (orspot diameter˜RH) for the humidity range of about 30-80% RH. Note thatthere can be minimal hysteresis observed upon raising and lowering thehumidity. This humidity dependency of DNA patterning generally points toa mechanism for transport of DNA from an AFM tip to a surface that isdependent on a water meniscus between the tip and substrate. Inaddition, a plot of spot size with respect to humidity can indicate thatthere is a minimum humidity at room temperature under which the DNAcannot be directly patterned via DPN printing at about 25° C. (forexample, x-intercept ˜27% RH at 23° C. as shown in the Working Examples,FIGS. 13 and 14). Finally, the fact that a linear regression line can bedrawn through points obtained under both high and low humidityconditions for experiments that were performed on different substratesand days underscores the control that humidity provides.

One of the advantageous properties inherent to DPN printing is thecapability of generating nanoscale patterns of multiple inks in highregistration. To demonstrate multi-nucleic acid ink capabilities, DPNprinting can be used to prepare a two-component nucleic acid array. Inorder to align patterns of two different thiol-modified DNA inks, C1 andC2, without cross-contamination, alignment markers can be first drawn attwo different locations on a gold substrate via DPN printing using, forexample, a thiol 16-mercaptohexadecanoic acid (MHA). Before patterning,the C1-coated tip can be used to image the MHA markers at low humidity(RH ˜25%, DNA does not transport under these conditions) in order tocalculate an offset coordinate relative to the MHA patterns. Next, thehumidity can be raised to, for example, 45% and a square array of dots(for example, diameter ˜760 nm), spaced for example about 2 μm apart canbe generated. Likewise, a second pattern, composed of C2 can bepositioned in alignment with the first by again imaging an MHA alignmentmarker with the coated tip at low humidity, calculating an offsetcoordinate, and then raising humidity and patterning, for example, atriangular array of 100 nm diameter dots. After patterning both nucleicacid inks, the unpatterned areas of the substrate can be passivated byODT treatment and imaged by tapping mode AFM. To verify the chemicalintegrity and activity of the patterns, the chip can be exposed to asolution of 13 nm G1-modified gold particles which were hybridized to L1(complementary to the C1 pattern) under hybridization conditions for 2h. The substrate can be then rinsed with PBS buffer with 0.025% SDS at45° C., and then exposed to 30 mm G1-modified particles which arehybridized to L2 (complementary to the C2 pattern). The particles canselectively assemble on the correct patterns with no evidence ofcross-contamination on the DNA spots or background. This embodiment notonly shows how nanoparticles can be used as diagnostic probes inAFM-based screening procedures, but also nanostructures fabricated viathe direct-write DPN printing approach can be used to control theassembly of nanoparticle-based architectures.

Often, DPN printing techniques can be done on gold substrates, which isin some cases undesirable from the standpoint of electronic and opticalmaterials applications. The gold-thiol system provides a useful methodfor patterning oligonucleotides using DPN printing. However, theelectrical conductivity of the gold substrate can prevent the study ofcharge transport and near-field optical phenomena in nanostructuresassembled on such surfaces, and furthermore can quench the emission fromany surface-bound fluorophores. To address these issues, and enableelectrical and optical characterization of the assembled nanostructures,DPN printing can be used to pattern nucleic acid on electricallyinsulating surfaces such as, for example, oxidized silicon wafers.

The surface of a thermally oxidized wafer can be activated by treatmentwith a functional silane coupling agent, such as, for example,3′-mercaptopropyltrimethoxysilane (MPTMS). The preparation and inking ofthe AFM tip can be performed as for the patterning of DNA onto goldsurfaces, however, oligonucleotides with 5′-terminal acrylamide groups(C3 and C4) can be used in place of oligonucleotides with terminalhexanethiol modifications. Under the DPN printing conditions of roomtemperature and 45% relative humidity, the acrylamide moieties can reactvia Michael addition with the pendant thiol groups of the MPTMS tocovalently link the nucleic acid to the surface. In addition, nucleicacid pattern formation on silicon oxide substrates shows a similartip-surface contact time dependence as for gold substrates. Followingpatterning, the substrate can be passivated by reaction with bufferedacrylic acid monomer at pH 10 (e.g., Apogent Discoveries thiol quenchbuffer). The biological activity of patterned C3 oligonucleotides can beverified by exposing the surface to a solution containing complementaryfluorophore-labeled DNA (L3F). The patterns can be subsequentlycharacterized by epi-fluorescence microscopy. DNA nanostructures onsilicon generated using this procedure can also be used to direct theassembly of complementary DNA-modified gold nanoparticles (modified withG2). With this technique, DNA spots on silicon oxide surfaces can begenerated and detected with diameters of ˜200 nm, nearly 10,000 timessmaller (in terms of areal density), than those in conventionalmicroarrays.

The nucleic acid which is subjected to direct write nanolithography isnot particularly limited. For example, the nucleic acid can besynthetically made, modified to include, for example, functional groupstailored for chemisorption or covalent bonding to the substrate, as wellas naturally occurring. It can be of low, medium, or high molecularweight, oligomeric or polymeric. It can be single-, double-, or eventriple-stranded. The nucleic acid can be based on deoxyribonucleic acid(DNA), ribonucleic acid (RNA), or combinations thereof. The structure ofnucleic acids is generally described in, for example, Calladine andDrew, Understanding DNA, The Molecule and How it Works, 2^(nd) Ed.,1997.

General types of nucleic acid which can be patterned by DPN printinginclude, for example, DNA, RNA, PNA, CNA, RNA, HNA, p-RNA,oligonucleotides, oligonucleotides of DNA, oligonucleotides of RNA,primers, A-DNA, B-DNA, Z-DNA, polynucleotides of DNA, polynucleotides ofRNA, T-junctions of nucleic acids, domains of non-nucleic acidpolymer-nucleic acid block copolymers and combinations thereof.Additional general types of nucleic acids include, for example, viralRNA or DNA, a gene associated with a disease, bacterial DNA, fungal DNA,nucleic acid from a biological source, nucleic acid which is a productof a polymerase chain reaction amplification, nucleic acid contactedwith nanoparticles, and nucleic acid double-stranded and hybridized withthe oligonucleotides on the nanoparticles resulting in the production ofa triple-stranded complex.

In general, the nucleic acid can be any of a group of organic substancesfound in cells and viruses that play a central role in the storage andreplication of hereditary information and in the expression of thisinformation through protein synthesis. Purines, pyrimidines,carbohydrates, and phosphoric acid generally characterize thefundamental organic substances of a nucleic acid. Purines andpyrimidines are nucleotides, a nucleoside in which the primary hydroxygroup of either 2-deoxy-D-ribose or of D-ribose is esterified byorthophosphoric acid. A nucleoside is a compound in which a purine orpyrimidine base is bound via a N-atom to C-1 replacing the hydroxy groupof either 2-deoxy-D-ribose or of D-ribose, but without any phosphategroups. The common nucleosides in biological systems are adenosine,guanosine, cytidine, and uridine (which contain ribose) anddeoxyadenosine, deoxyguanosine, deoxycytidine and thymidine (whichcontain deoxyribose). Thus, a purine base may be an adenine nucleotideor a guanine nucleotide. A pyrimidine base may be thymine nucleotide, acytosine nucleotide, or a uracil nucleotide.

The sequence of a nucleic acid may be random or specific so as to encodea desired amino acid structure. For instance, a group of threenucleotides may comprise a codon. One codon comprises an amino acid. Thecoding region of a nucleic acid comprises codons.

The nucleic acid can exist freely, or can be bound to peptides orproteins to form nucleoproteins in discreet bundles or structured formssuch as, for example, chromosomes. A nucleic acid also can exist insingle-stranded or double-stranded forms. A nucleic acid may also belinear, circular, or supercoiled. Nucleic acid may be isolated directlyfrom a cell or organelle. A plasmid or cloning vector are also examplesof nucleic acids.

The nucleic acid can be made up of nucleotides, each containing acarbohydrate sugar (deoxyribose), a phosphate group, and mixtures ofnitrogenous purine- and pyrimidine-bases. The sugar may be of a cyclicor acyclic form. DNA comprises only thymine and cytosine pyrimidines andno uracil. DNA may be isolated from a cell as genomic, nuclear, ormitochondrial DNA, or made synthetically, i.e., by chemical processes.

A gene present in a cell typically comprises genomic DNA made up ofexonic and intronic stretches of DNA. The exonic stretches comprisesnucleotides that comprise codons that encode amino acids, whereas theintronic stretches of DNA comprise nucleotides that likely do notcomprise codons that encode amino acids. The nucleotide sequence ofpurines and pyrimidines determine the sequences of amino acids in thepolypeptide chain of the protein specified by that gene.

DNA may also be isolated as complementary or copy DNA (cDNA) producedfrom an RNA template by the action of RNA-dependent DNA polymerase. Forexample, the cDNA can be about 100-800mer strands from PCRamplification. If the RNA template has been processed to remove introns,the cDNA will not be identical to the gene from which the RNA wastranscribed. Thus, cDNA may comprise a stretch of nucleotides that arelargely exonic in nature.

When in double-stranded form, the two DNA strands form a double helix.In this helix, each nucleotide in one strand is hydrogen bonded to aspecific nucleotide on the other strand. Thus, in DNA, adenine bondswith thymine and guanine bonds with cytosine. The ability of nucleotidespresent in each strand to bind to each other determines that the strandswill be complementary, e.g., that for every adenine on one strand therewill be a thymine on the other strand.

RNA can be generally similar to DNA, but contains the sugar riboseinstead of deoxyribose and the base uracil instead of thymine. RNA canbe single-stranded or double-stranded and is transcribed from a cell'sDNA. An RNA molecule may form a hairpin loop or other double-strandedstructures. RNA may be template RNA, messenger RNA (mRNA), total RNA, ortransfer RNA (tRNA). polysome. RNA-DNA hybrid molecules can be depositedaccording to the present invention. Furthermore, protein-nucleic acids,or “peptide nucleic acids” (“PNA”) also may be used in accordance withthe present invention.

The binding properties exhibited between complementary nucleotides makesnucleic acids useful as probes that can bind to other nucleic acids.Nucleic acids can be labelled and used as probes. By any one of a numberof standard labelling techniques, nucleic acid probes can be used todetect, by hybridization, another nucleic acid. That hybridization canbe visualized or detected if the label is, for example, a fluorescent,radioactive, or enzymatic label. Thus, a nucleic acid of the presentinvention also can be labelled, or modified so as to comprise adetectable entity, like a fluorescent marker or tag, a gold particle,streptavidin, digoxigenin, a magnetic bead, or other markers known tothe skilled artisan. See, for example, U.S. Pat. No. 4,626,501 (“LabeledDNA”) to Landes, which is hereby incorporated by reference.

Nucleotides and nucleic acids also can be modified so that it isprotected against nucleic acid degradation. For instance, a nucleic acidmay be encapsulated within a liposome. Alternatively, a thiol group maybe incorporated into a polynucleotide, such as into an RNA or DNAmolecule, by replacing the phosphorous group of the nucleotide. When soincorporated into the “backbone” of a nucleic acid, a thiol can preventcleavage of the DNA at that site and, thus, improve the stability of thenucleic acid molecule.

U.S. Pat. No. 5,965,721 to Cook et al. is also incorporated byreference, disclosing oligonucleotides which can be patterned and canhave improved nuclease resistance and improved cellular uptake.

Thus, the bioavailability of a nucleic acid treatment in vivo may beimproved by modifying the nucleic acid as described. For instance, amodified nucleic acid formulation may have an increased half-life and/orbe retained in plasma for longer periods of time than non-modifiednucleic acids. A formulation of nucleic acid and polyethylene glycol,for instance, may also increase the half-life of the nucleic acid invivo, as could any known slow-release nucleic acid formulation. Thus,modifying a nucleic acid may increase the effectiveness of the nucleicacid in vivo and/or its bioavailability.

The size of a nucleic acid can range considerably, from the size of afew nucleotides, to an oligonucleotide, or probe, to a polynucleotide,gene, chromosome fragment to entire chromosomes and genomes. Forinstance, a single- or double-stranded nucleic acid may be at least 10-,20-, 30-, 40-, 50-, 60-, 70-, 80-, 90, or 100-nucleotides or base pairs(bp) in length. Larger still, a nucleic acid may be at least 0.2 kb, 0.3kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, or 1.0 kb in size.Indeed, a nucleic acid for use in the present invention can be at least1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, or 10 kb or largerin size. One preferred size range is 1-2 kb. The nucleic acid can be achain of varying length of nucleotides and are typically calledpolynucleotides or oligonucleotides. An oligonucleotide is an oligomergenerally resulting from a linear sequences of nucleotides. Theoligonucleotide can comprise, for example, about 2 to about 100, about 2to about 20, about 10 to about 90, or about 15 to about 35 nucleotides.In oligonucleotide arrays, about 25-mer oligonucleotides can be used.Another particular range is about 60- to about 80-mers, which arerelatively long oligonucleotides.

Microarray methods, including selection of nucleic acid, probling,labeling, and detection, are described in U.S. Pat. Nos. 6,379,932 and6,410,231 (Incyte Genomics) and can be used. These patents areincorporated by reference in their entirety. Although these referencesmention dip pen nanolithographic methods, they do not suggest how orprovide guidance on how dip pen nanolithographic methods can be used tomake improved nanoarrays as described herein.

A compound comprising a single nucleotide can also be used as ink.Mixtures of nucleic acids can be used, and different spots on an arraycan comprise different nucleic acids.

A nucleic acid for deposition according to the present invention may beformulated or mixed with other elements prior to, or after direct writedeposition onto a substrate surface. Thus, an “ink” of the presentinvention may comprise other chemicals, compounds, or compositions fordeposition onto a substrate surface in addition to a desired nucleicacid sample. As described above, solvent and salt can be used to applythe nucleic acid to the tips. Surfactants can be used. For instance,proteins, polypeptides, and peptides may be deposited along with adesired nucleic acid onto a substrate surface.

Nucleic acid arrays, and the types of nucleic acids used therein, aredescribed for example in A Primer of Genome Science, G. Gibson and S.Muse, 2002, Chapters 3-4 (pages 123-181), which is hereby incorporatedby reference. This reference, for example, describes both cDNAmicroarrays and oligonucleotide arrays, labeling, hybridization, andstatistical analysis. cDNA arrays can be used for monitoring therelative levels of expression of thousands of genes simultaneously.PCR-amplified cDNA fragments (ESTs) can be spotted and probed againstfluorescently or radioactively labeled cDNA. The intensity of the signalobserved can be assumed to be in proportion to the amount of transcriptpresent in the RNA population being studied. Differences in intensityreflect differences in transcript level between treatments. Statisticaland bioinformatic analyses can then be performed, usually with the goalof generating hypotheses that may be tested with established molecularbiological approaches. Current cDNA microarrays, however, can have anupper limit of 15,000 elements and are unable to represent the completeset of genes present in higher eukaryotic genomes. The advantages anddisadvantages of oligonucleotide versus cDNA microarrays are describedin the aforementioned A Primer of Genome Science and can be used inconstructing nucleic acid nanoarrays as described herein.

DIP PEN nanolithographic printing, particularly parallel DIP PENnanolithographic printing, is useful for the preparation of nucleic acidnanoarrays, particular combinatorial nanoarrays. An array is anarrangement of a plurality of discrete sample areas, or pattern units,forming a larger pattern on a substrate. The sample areas, or patterns,may be any shape (e.g., dots, lines, circles, squares or triangles) andmay be arranged in any larger pattern (e.g., rows and columns, lattices,grids, etc. of discrete sample areas). Pattern can refer to anindividual unit or a larger collection of individual units. Each samplearea may contain the same or a different sample as contained in theother sample areas of the array. A “combinatorial array” is an arraywherein each sample area or a small group of replicate sample areas(usually 2-4) contain(s) a sample which is different than that found inother sample areas of the array. A “sample” is a material or combinationof materials to be studied, identified, reacted, etc.

DIP PEN nanolithographic printing, particularly parallel DIP PENnanolithographic printing, is particularly useful for the preparation ofnanoarrays and combinatorial nanoarrays on the submicrometer scale. Anarray on the submicrometer scale, or nanoscale, means that at least oneof the lateral dimensions (e.g., length, width or diameter) of thesample areas, excluding the depth, is less than 1 μm. These dimensionsare lateral dimensions, generally in the plane of the substrate. DIP PENnanolithographic printing, for example, can be used to prepare dots thatare about 10 nm in diameter. With improvements in tips (e.g., sharpertips), dots can be produced that approach 1 nm in diameter. Arrays on asubmicrometer scale allow for faster reaction times and the use of lessreagents than the currently-used microscale (i.e., having dimensions,other than depth, which are 1-999 μm) and larger arrays. Also, moreinformation can be gained per unit area (i.e., the arrays are more densethan the currently-used micrometer scale arrays). Finally, the use ofsubmicrometer arrays provides new opportunities for screening. Forinstance, such arrays can be screened with SPM's to look for physicalchanges in the patterns (e.g., shape, stickiness, height) and/or toidentify chemicals present in the sample areas, including sequencing ofnucleic acids.

Each sample area of an array can contain a single sample or a singledeposit. For instance, the sample may be a biological material, such asa nucleic acid as described extensively above (e.g., an oligonucleotide,DNA, or RNA), protein or peptide (e.g., an antibody or an enzyme),ligand (e.g., an antigen, enzyme substrate, receptor or the ligand for areceptor), or a combination or mixture of biological materials (e.g., amixture of proteins or nucleic acids).

The present invention is particularly focused on nucleic acid,oligonucleotide, and DNA nanoarrays. Arrays and methods of using arraysare known in the art. For instance, such arrays can be used forbiological and chemical screenings to identify and/or quantitate abiological or chemical material (e.g., immunoassays, enzyme activityassays, genomics, and proteomics). Biological and chemical libraries ofnaturally-occurring or synthetic compounds and other materials,including cells, can be used, e.g., to identify and design or refinedrug candidates, enzyme inhibitors, ligands for receptors, and receptorsfor ligands, and in genomics and proteomics. Arrays of microparticlesand nanoparticles can be used for a variety of purposes (see for exampleExample 7 of US Patent Publication 2002/0063212 A1). Arrays can also beused for studies of crystallization, etching (see for example Example 5of US Patent Publication 2002/0063212 A1) and the like. Referencesdescribing combinatorial arrays and other arrays and their uses include,for example, U.S. Pat. Nos. 5,747,334, 5,962,736, and 5,985,356, and PCTapplications WO 96/31625, WO 99/31267, WO 00/04382, WO 00/04389, WO00/04390, WO 00/36 136, and WO 00/46406, which are hereby incorporatedby reference in their entirety. Finally, results of experimentsperformed on the arrays of the invention can be detected by conventionalmeans (e.g., fluorescence, chemiluminescence, bioluminescence, andradioactivity). Alternatively, an SPM can be used for screening arrays.For instance, an AFM can be used for quantitative imaging andidentification of molecules, including the imaging and identification ofchemical and biological molecules through the use of an SPM tip coatedwith a chemical or biomolecular identifier. See, for example, Frisbie etal., Science, 265, 2071 2074 (1994); Wilbur et al., Langmuir, 11,825-831 (1995); Noy et al., J. Am. Chem. Soc., 117, 7943-7951 (1995);Noy et al., Langmuir, 14, 1508-1511 (1998); and U.S. Pat. Nos.5,363,697, 5,372,93, 5,472,881 and 5,874,668, the complete disclosuresof which are incorporated herein by reference.

Direct-write nanolithographic printing is particularly useful for thepreparation of nucleic acid nanoarrays, arrays on the submicrometerscale having nanoscopic features, wherein a plurality of dots or aplurality of lines can be formed on a substrate. The plurality of dotscan be a lattice of dots including hexagonal or square lattices as knownin the art. The plurality of lines can form a grid, includingperpendicular and parallel arrangements of the lines.

The lateral dimensions of the individual patterns including dotdiameters and the line widths can be of a nanoscale, for example, about1,000 nm or less, about 500 nm or less, about 300 nm or less, about 200nm or less, and more particularly about 100 nm or less. The range indimension can be for example about 1 nm to about 750 nm, about 10 nm toabout 500 nm, and more particularly about 100 nm to about 350 nm. Asmall range of about 10 nm to about 100 nm can be used.

The number of patterns in the plurality of patterns is not particularlylimited for a single substrate and generally high pattern density isdesired. It can be, for example, at least 10, at least 100, at least1,000, at least 10,000, even at least 100,000. Square arrangements arepossible such as, for example, a 10×10 array. Higher density arrays arepreferred, generally at least 100, preferably at least 1,000, morepreferably, at least 10,000, and even more preferably, at least 100,000discrete elements per square centimeter. Remarkably, the nanotechnologydescribed herein can be used to generate ultra-high density nanoarrayscomprising more than one million, more than 100,000,000, and moreparticularly, even more than one billion, discrete elements per squarecentimeter as a pattern density.

The distance between the individual patterns on the nanoarray can varyand is not particularly limited. For example, the patterns can beseparated by distances of less than one micron or more than one micron.The distance can be, for example, about 300 to about 1,500 microns, orabout 500 microns to about 1,000 microns. Distance between separatedpatterns can be measured from the center of the pattern such as thecenter of a dot or the middle of a line.

The amount of nucleic acid in a particular spot or deposit is notlimited but can be, for example at a pg or ng level including, forexample, about 0.1 ng to about 100 ng, and more particularly, about 1 ngto about 50 ng. Spotting solution methods used in nucleic acidmicroarray technology can be also used as desired in nanoarraytechnology.

By methods described herein, a nucleic acid nanoarray can be preparedcomprising a substrate and a plurality of patterns of nucleic acid onthe substrate, wherein the patterns of nucleic acid are chemisorbed orcovalently bonded to the substrate, have lateral dimensions of about1,000 nm or less and are separated from each other by distances of 1,000nm or less, and are hybridizable to complementary nucleic acids. In apreferred embodiment, the nucleic acid nanoarray comprises at least1,000 patterns of nucleic acid, wherein the lateral dimensions of thepatterns are about 500 nm or less, the patterns are separated from eachother by distances of 500 nm or less, and the patterns are in the formof dots. In another preferred embodiment, the nucleic acid nanoarraycomprises at least 10,000 patterns of nucleic acid, wherein the lateraldimensions of the patterns are about 200 nm or less, the patterns areseparated from each other by distances of 500 nm or less, and thepatterns are in the form of dots.

Conventional substrates known for use with direct-write nanolithographyand nucleic acid microarrays including glass can be used. In addition tothose described above, substrates include membranes, plastic orpolymeric gels, microwells, electrodes, nanogaps, and sensor devices.Membranes include those used in current nucleic acid microarraytechnology including nitrocellulose and nylon membranes. Substrates canbe treated with, for example, a monolayer or a primer layer before thenucleic acid is deposited onto the substrate. The primer layer can bedesigned to covalently anchor the nucleic acid. For example, if5′-aminoacylated PCR primers are used to amplify ESTs, aldehyde-basedcoatings can be used that link to the end of the nucleic acid molecules.Multi-layer architectures can be made.

In view of the rapid proliferation of bioconjugated nanoparticle labelsand building blocks, the method described here should allow the DPN- andnucleic acid-templated assembly of a wide variety of metallic,semi-conducting, magnetic, and insulating nanostructures, on bothmetallic and insulating substrates. Exemplary publications include (1)M. Bruchez et al, Science, 281, 2013 (1998), (2) S. R. Nicewarner-Penaet al., Science, 294, 137 (2001); (3) Y. Cui, et al., Science, 293, 1289(2001). These structures can in turn be used to address issues inmolecular electronics, photonics, high-density information storage, andbiosensing. The method also suggests new routes for investigating thefundamental limits of microarray miniaturization. With the resolutiondemonstrated here, arrays with ˜100,000 nucleic acid or oligonucleotidespots could be generated in an area the size of a typical AFM scanner(100 μm by 100 μm) on time scales comparable with those of conventionalrobotic spotting methods, thereby making possible the investigation ofscanned probe methods of nano- and microarray fabrication and readout.Robot methods, for example, can be limited by spotting rates of only afew deposits per second, which means it can take up to two days forrobots to prepare 100 microarrays containing at least 5000 clones.

Nanoparticles useful in the practice of the invention include metal(e.g., gold, silver, copper, and platinum), semiconductor (e.g., CdSe,CdS, and CdSe or CdS coated with ZnS) and magnetic (e.g.,ferromagnetite), colloidal materials. Other nanoparticles useful in thepractice of the invention include ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS,PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₂As2, InAs, and GaAs. The sizeof the nanoparticles is preferably from about 5 nm to about 150 nm (meandiameter), more preferably, from about 5 nm to about 50 nm, and morepreferably, about 10 nm to about 30 nm. Methods of making and usingnanoparticles bonded to nucleic acids are described in U.S. Pat. No.6,361,944, which is hereby incorporated by reference.

The invention is further illustrated by the following working examples,which do not limit the scope of the invention as described in detailabove and as claimed below.

WORKING EXAMPLES

The invention is further illustrated by the following non-limitingExperimental Section and Working Examples. In addition, Working Examples1-7 in U.S. patent publication 2002/0063212 A1 to Mirkin et al.illustrate various embodiments for DIP PEN™ nanolithographic printing,and are hereby incorporated by reference.

Experimental Section

General methods and materials. 1-Octadecanethiol was purchased fromAldrich (Milwaukee, Wis.). Thiol-modified 15-mer DNA with apolyethyleneglycol spacer (PEG₆) corresponding to 18 atoms between theanchor groups and DNA (C1, C2, see Table I) was purchased from IDT(Coralville, Iowa, PAGE purified) and used without further purification.Linking DNA (L1 and L2) and gold nanoparticle-bound DNA (G1) used ingold substrate patterning experiments were synthesized as previouslyreported (see, for example, J. J. Storhoff et al., J. Am. Chem. Soc.,120, 1959 (1998); and U.S. Pat. Nos. 6,417,340 and 6,361,944 to Mirkinet al.).

Acrylamide modified (Acrydite™) 12- and 15-mer DNA (C3 and C4) werepurchased from IDT (RP HPLC purified). Linking DNA (L3 and L4) andnanoparticle-bound DNA (G2 and G3) used in silicon oxide patterningexperiments were synthesized by known methods (see, for example, J. J.Storhoff et al., J. Am. Chem. Soc., 120, 1959 (1998); and U.S. Pat. Nos.6,417,340 and 6,361,944 to Mirkin et al.).

TABLE I Oligonucleotide sequences used for DPN patterning, nanoparticlemodification, and linking of particles to DNA-patterned surfaces. SEQStrand ID Name Sequence and End Modifications NO: C1 5′HS(CH₂)₆-PEG₆-GAGGGA TTA TTG TTA 1 C2 5′HS(CH₂)₆-PEG₆-AGT CGC TTC TAC CAT 2 L1 5′AGA GTTGAG CTA TAA CAA TAA TCC CTC 3 L2 5′AGA GTT GAG CTA ATG GTA GAA GCG ACT 4G1 5′TAG CTC AAC TCT A₂₀(CH₂)₃SH 5 C3 5′Acrydite-PEG₆-ATC CTT ATC AATATT 6 C4 5′Acrydite-PEG₆-CGC ATT CAG GAT 7 L3 5′GGA TTA TTG TTA AAT ATTGAT AAG GAT 8 L4 5′TAC GAG TTG AGA ATC CTG AAT GCG 9 L3F OregonGreen488X-5′GGA TTA TTG TTA AAT 10 L4F Oregon Green488X-5′TAC GAG TTGAGA 11 G2 5′TCT CAA CTC GTA A₁₀(CH₂)₃SH 12 G3 5′TAA CAA TAA TCCA₁₀(CH₂)₃SH 13Gold thin films on single crystalline silicon wafers were preparedaccording to previously reported procedures (see L. M. Demers et al.,Anal. Chem. 72, 5535 (2000). Silicon oxide substrates were cleaned inpiranha solution (3:1 sulfuric acid to 30% hydrogen peroxide) prior tosilanization with mercaptopropyltrimethoxysilane (MPTMS) for 2 h byvapor transport (see for example D. G. Kurth et al. Langmuir, 9, 2965(1993), after which they were rinsed with ethanol and cured underflowing N₂ for 10 min at 80° C.

AFM tip preparation. Conventional silicon nitride probe chips (springconstant ˜0.3 Nm⁻¹, Thermomicroscopes, Sunnyvale, Calif.) were firstcleaned for 10 min in piranha solution (1H₂O₂: 3H₂SO₄), rinsed with DIwater, ethanol, and toluene, and then immersed in a 1% vol/vol solutionof 3-aminopropyltrimethoxysilane (APS) in toluene in a glass petri dishfor 1-2 h. After silanization the chips were rinsed with toluene, andthen dried under a stream of nitrogen. In a typical procedure, tips werecoated immediately with thiol-modified DNA by dipping in tip coatingsolution (1 mM DNA, 90% dimethylformamide (dmf), 10% water, 0.3 M MgCl₂)for 20 s and subsequently drying with compressed difluoroethane.Amine-coated tips were well coated with dimethylformamide (DMF)solutions of DNA if dipped immediately into the solution aftersilanization, but in general only poorly coated after 12 h, presumablydue to contamination of the tip surface.

Dip-Pen Nanolithographic Printing experiments. Dip-pen nanolithographicprinting was performed using DNA-coated AFM tips (contact force ˜2.5 nN)in a controlled atmosphere glovebox. The glovebox enabled control overtemperature and humidity to within ±5° C. and ±5% relative humidity. AllDPN printing experiments were performed using a Park ScientificInstruments Autoprobe CP AFM with a customized DPN printing softwareinterface.

Gold nanoparticle assembly. Gold nanoparticles were modified withthiol-functionalized DNA by known methods (see, for example, J. J.Storhoff et al., J. Am. Chem. Soc., 120, 1959 (1998); and U.S. Pat. Nos.6,417,340 and 6,361,944 to Mirkin et al.). For assembly onto patternedgold substrates, nanoparticles (20 μl, 13 nm diameter, 5 nM) modifiedwith thiol-capped DNA were hybridized overnight to linking DNA for atleast 12 h in hybridization solution (final concentrations 0.1 μM DNA,0.3 M PBS (0.01 M phosphate buffer pH 7, 0.3 M NaCl), 0.025% sodiumdodecyl sulfate (SDS)). The particle-DNA solution was then placed as adroplet on the horizontal patterned substrate and incubated at roomtemperature for 3 h. Following incubation, the slide was placed in apolypropylene tube and rinsed under a stream of buffer at roomtemperature (0.3 M PBS, 0.025% SDS), followed by 0.3 M ammonium acetate,pH 7, to remove salt from the surface to prepare the slide for AFMimaging. For particle assembly onto DNA-patterned SiO_(x) substrates,linker DNA was hybridized to DNA-modified nanoparticles (0.5 μM linker,10 nM particles, total volume 0.2 ml) by heating to 60° C. for 5 min,then allowing to cool for 30 min. The solution was subsequently dilutedto 1 ml total volume with 0.3 M PBS 0.025% SDS and used forhybridization to patterned substrates as described above.

Imaging experiments. Tapping mode topography and phase AFM images of DNApatterns and nanoparticles were obtained using a Digital Instruments(Santa Barbara, Calif.) Nanoscope IIIa with silicon cantilevers fromDigital Instruments, spring constant ˜40 Nm⁻¹. All AFM images wereprocessed only by applying first or second order flattening function.For fluorescence imaging the patterned substrates were hybridized atroom temperature with fluorophore-labeled, complementary DNA for 30 min.in 2×SSPE buffer (2 μM DNA, 0.3 M NaCl, 0.02 M sodium phosphate, 0.002 MEDTA, 0.2% SDS, pH 7.4), rinsed with 2×SSPE 0.2% SDS and then immersedin 2×SSPE 0.2% SDS for 10 min. The substrates were rinsed with 0.3 Mammonium acetate and blown dry with N₂ prior to fluorescence imagingwith a Zeiss Axiovert 100 microscope with a Hg lamp white lightexcitation source.

Example 1 Patterning of Nucleic Acid by Dip Pen NanolithographicPrinting

This example describes a method of patterning nucleic acids by DPNprinting which provides improvements in tip-coating, diffusion to thesubstrate surface, and pattern stability during post-processing steps.This method employs an aminosilane-modified silicon nitride AFMcantilever-tip assembly and cyclic disulfide and polyethyleneglycolmodified synthetic nucleic acid to form stable oligonucleotide patternson a gold substrate, which can be hybridized with probes including, forexample, nanoparticle probes. Judicious selection of relative humidityduring printing provides for control of the size of the printedfeatures.

1. AFM Tip Pretreatment Procedure

A conventional silicon nitride AFM tip was first modified withtrimethoxyamino-propylsilane (APS). The cantilevers were cleaned inpiranha etch (3:1 concentrated sulfuric acid/30% hydrogen peroxide) for10 minutes, then rinsed in Nanopure water, and ethanol and dried in astream of nitrogen. Tips were then covalently modified by immersion in1% solution of APS in toluene for 1 hour, and then rinsed with toluene.This procedure created a positively-charged surface to which thenegatively-charged nucleic acids adhered.

2. AFM Tip Coating

A 1 mM solution of modified synthetic DNA, containing (a) either asingle hexanethiol moiety or a cyclic disulfide epiandrosterone linkerand (b) a polyethylene glycol (PEG) spacer (see FIGS. 1 and 3), wasprepared in 20 microliters of dimethylformamide (DMF) and 1 microliterof 1.5 M MgCl₂ in water. Pretreated AFM tips were coated with DNA bydipping the tips in the solution for 20-60 seconds (s) and blowing drywith compressed air or nitrogen. In general, the DMF plays a role insolubilizing the DNA and can allow wetting of the APM tip with thecoating solution. In general, the low concentration of MgCl₂ was used toachieve high density DNA patterns on a surface by screening repulsiveinteractions between DNA strands. In general, the PEG spacer can beimportant in providing diffusion of the DNA to the substrate.

3. Direct Patterning of DNA via DPN Printing

To transfer DNA to a gold substrate, the DNA-coated AFM tip waspositioned relative to the substrate and brought close to the substrateso as to be in contact with the substrate (see FIG. 3). Tip movementalong the surface was controlled by DPN printing software. Control overpattern size was achieved by varying the relative humidity in anatmospheric control chamber. For example, the diameter of dot patternscould be varied from about 780 nm at about 88% relative humidity (upperrow) to about 430 nm at about 50% relative humidity (lower row; see FIG.4—All dots were made by holding the AFM tip in position for 20 seconds.)The disulfide linker forms a chelate bond to the gold surface whichincreases the stability of the DNA binding to the surface. DNA bound inthis way resists displacement by alkanethiols (such as1-octadecanethiol) which are used for passivation of the surroundingunpatterned gold. The DNA patterns were shown to have biologicalactivity by hybridization to gold nanoparticle probes which had beenmodified with complementary DNA sequences (see FIG. 5 and see, forexample, Letsinger, R. L, Elghanian, R.; Viswanadham, G.; Mirkin, C. A.Bioconjugate Chemistry, 2000, 11, 289-291; and PCT application WO98/04740.

Examples 2 and 3

The use of direct-write dip-pen nanolithography printing (e.g., DPNprinting) to generate covalently anchored, nanoscale patterns ofoligonucleotides on metallic, conductive substrates such as gold (Ex. 2)and insulating substrates (Ex. 3) was carried out. Modification of DNAwith hexanethiol groups provided for patterning on gold (Ex. 2), andoligonucleotides bearing 5′-terminal acrylamide groups were patterned onderivatized silica (Ex. 3). Feature sizes ranging from many micrometersto less than 100 nanometers were achieved in these examples, and theresulting patterns exhibited the sequence-specific binding properties ofthe DNA from which they were composed. The patterns were used to directthe assembly of individual oligonucleotide-modified particles on asurface, and the deposition of multiple DNA sequences in a single arraywas demonstrated.

DPN printing was used to pattern oligonucleotides on gold (Ex. 2) andsilicon oxide (Ex. 3) surfaces. Several keys were identified thatfacilitated DNA patterning. First, the AFM tip was well coated with DNA.Although unmodified silicon nitride cantilevers have been used todeposit a variety of hydrophobic molecules by DPN, such cantilevers canyield DNA patterns with feature sizes and shapes that at times arecontrolled with difficulty. Improved control over DNA patterning wasachieved through surface modification of a silicon nitride AFMcantilever with 3′-aminopropyltrimethoxysilane (for 1 hour in a 1% v/vsolution in toluene), which promoted reliable adhesion of the DNA ink tothe tip surface.

The silanized tips were coated with DNA by dipping them for 10 s into a90% dimethylformamide/10% water solution containing 1 mM DNA and 0.3 MMgCl₂ and then the tips were blown dry with compressed difluoroethane.The positively charged hydrophilic tip surfaces were readily wetted bythis DNA ink solution, and these AFM tips could be used in DPN printingexperiments for several hours before they were recoated. The tips werealso successfully used by coating them with an evaporated gold layer anda self-assembled monolayer of cysteamine. Furthermore, it was found thatcontrol of ambient humidity enabled reliable DPN patterning ofoligonucleotides. Unless specifically noted, all patterning wasperformed in an environmentally controlled glovebox at a relativehumidity of 45±5% at 23±3° C.

The judicious choice of an ink-substrate combination also facilitatedthe DPN printing process. In Example 2, for example, hexanethiolmodified oligonucleotides were used to directly pattern gold substrateswith features ranging from 50 nm to several micrometers in size (FIGS.6-10). It is generally believed that the hexanethiol group of the DNAchemisorbs to the underlying Au surface (see, for example, T. M. Herneet al., J. Am. Chem. Soc., 119, 8916 (1997). After the substrates werepatterned with oligonucleotides, they were immersed in an ethanolsolution of 1-octadecanethiol (ODT, 1 mM) for 1 minute. This procedurecoated the unpatterned gold surface with a hydrophobic monolayer,passivating it toward the nonspecific adsorption of DNA, or DNA-modifiednanoparticles, in subsequent hybridization experiments. After the ODTtreatment of the substrate, the oligonucleotide patterns were imaged bytapping-mode AFM and exhibited feature heights of 2 to 5 nm (FIG. 6A).See, R. Levicky et al. J. Am. Chem. Soc., 120, 9787 (1998). Theimmobilized DNA retained its highly specific recognition properties, andthe patterns could be used to direct the assembly of 13-nm-diameteroligonucleotide-modified gold nanoparticles (FIG. 6B). Structures werefabricated by this process on the many micrometer to sub-100-nm-lengthscale, and individual particles were placed on a surface in the form ofa preconceived architecture (FIGS. 6-9). The interactions between theDNA nanopatterns and the oligonucleotide-modified nanoparticles werehighly selective; in the absence of a complementary linking strand,there was almost no nonspecific binding (FIG. 7).

Although the gold-thiol system provided an excellent method forpatterning oligonucleotides using DPN printing, the electricalconductivity of the gold substrate prevents the study of chargetransport and near-field optical phenomena in nanostructures assembledon such surfaces and also quenches the emission from any surface-boundfluorophores. Thus, DPN printing methods were developed to generatepatterns of DNA on oxidized silicon wafers (FIG. 10). The surface of athermally oxidized wafer was activated by treatment with3′-mercaptopropyltrimethoxysilane (MPTMS). See, D. G. Kurth et al.,Langmuir, 9, 2965 (1993). The preparation and inking of the AFM tip wereperformed substantially the same as for the patterning of DNA onto goldsurfaces, but in this case, oligonucleotides with 5′-terminal acrylamidegroups were used. See, M. Kenney, Biotechniques, 25, 516 (1998). Underthe DPN printing conditions of room temperature and 45% relativehumidity, the acrylamide moieties react by Michael addition with thependant thiol groups of the MPTMS to covalently link the DNA to thesurface (see FIG. 11). After patterning, the substrate was passivated byreaction with buffered acrylic acid monomer at pH 10 (ApogentDiscoveries Quench Solution, 30 mm.). After all DNA spots and sequenceshave been patterned, the substrate was typically left overnight to allowthe formation of the thioether adduct before the substrate was washedand the unreacted thiol groups in the unpatterned areas are quenched.The biological activity of the patterned oligonucleotides was verifiedby exposing the surface to a solution containing both complementary andnoncomplementary fluorophore-labeled DNA. The patterns were subsequentlycharacterized by epifluorescence microscopy (FIG. 10A). In all cases,only fluorescence corresponding to the complementary target and thepatterned area was detected. The same DNA nanostructures [afterdehybridization of the single-stranded complement by rinsing withdeionized (Dl) water] could be used to direct the assembly ofcomplementary DNA-modified gold nanoparticles (FIG. 10B). With thistechnique. DNA spots were generated and detected with diameters of ˜50nm, nearly 160,000 times smaller (in terms of areal density) than thosein conventional microarrays.

Example 4

An important feature of DPN printing is the ability to generate patternsof specific chemical functionality over a large range of length scaleswhile exhibiting control over feature size in a preconceived manner.Surprisingly, patterns of highly charged macromolecules such asoligonucleotides can be transferred to a substrate in much the same wayas can small hydrophobic molecules. On both gold and MPTMS-modifiedsilicon oxide substrates, the transport of DNA from the AFM tip to thesurface followed the same linear increase in pattern area with contacttime predicted theoretically (see, J. Jang et al, J. Chem. Phys., 1115,2721 (2001)), as well as observed for alkanethiols on gold (see D. A.Weinberger et al., Adv. Mater., 12, 1600 (2000) and for silazanes onsilicon and gallium arsenide (see, Maynor et al., Langmuir, 17, 2575(2001), (FIGS. 12 and 13A,B). Although the rate constants can bedifferent for each ink-substrate pair, this result underscores thecontrol DPN printing offers for patterning compounds ranging from smallmolecules and salts to organic macromolecules on a variety ofsubstrates.

Example 5

On both gold and silicon oxide, the transport rate and pattern size ofthe DNA can be tailored with careful humidity control. It is thuspossible to vary feature size over a large dynamic range on a reasonabletime scale. For example, on gold, the diameter of a spot created byholding the AFM tip for 10 s changes from 50 to 300 nm with a relativehumidity change of 15% (FIG. 13C; see also FIG. 14). This humiditydependence generally points to a mechanism for transport of DNA from anAFM tip to a surface, which is generally dependent on the water meniscusbetween the tip and substrate. See, for example, Piner et al., Langmuir,15, 5457 (1999).

Example 6

To demonstrate multi-DNA ink capabilities, DPN printing was used toprepare a two-component DNA array on an oxidized silicon substrate andverified its sequence-specific activity by hybridization withcomplementary fluorophore-labeled probes (FIGS. 15 and 16). To furtherverify the chemical integrity of the patterns, the same chip was treatedwith DI water to remove the fluorophore-labeled DNA and then exposed toa solution containing a mixture of 5- and 13-nm-diameter goldnanoparticles. The large and small particles were modified with DNAcomplementary to the first and second patterns, respectively. Theparticles selectively assembled on the correct patterns underappropriate hybridization conditions. This experiment not only shows hownanoparticles can be used as diagnostic probes in AFM-based screeningprocedures but also shows how nanostructures can be formed by thedirect-write DPN approach to control and fabricate the assembly ofnanoparticle-based architectures.

1. A method for depositing nucleic acid onto a substrate by direct-writenanolithography comprising the step of: positioning at least onenanoscopic tip relative to a substrate so that the tip and substrateapproach each other, and wherein nucleic acid is transferred from thetip to the substrate to generate a stable nucleic acid nanoscale patternon the substrate which is hybridizable with complementary nucleic acid.2. The method according to claim 1, wherein the nanoscopic tip is ascanning probe microscopic tip.
 3. The method according to claim 1,wherein the nanoscopic tip is an atomic force microscopic tip.
 4. Themethod according to claim 1, wherein the scanning probe microscopic tipis a hollow tip.
 5. The method according to claim 1, wherein the tip ismodified to have a positive charge.
 6. The method according to claim 1,wherein the tip is modified to have a positive charge of amino groups.7. The method according to claim 1, wherein the tip is an atomic forcemicroscope tip which has been coated with a solution comprising nucleicacid and salt.
 8. The method according to claim 1, wherein the nucleicacid comprises deoxyribose nucleic acid.
 9. The method according toclaim 1, wherein the nucleic acid is single-strand nucleic acid.
 10. Themethod according to claim 1, wherein the nucleic acid comprisesfunctional groups to provide for chemisorption or covalent bonding tothe substrate.
 11. The method according to claim 1, wherein the nucleicacid is modified with a spacer which separates the nucleic acid from afunctional group which provides for chemisorption or covalent bonding tothe substrate.
 12. The method according to claim 1, wherein the nucleicacid is an oligonucleotide.
 13. The method according to claim 1, whereinthe substrate is an electrical conductor.
 14. The method according toclaim 1, wherein the substrate is an electrical insulator.
 15. Themethod according to claim 1, wherein the transfer is carried out atrelative humidity of at least about 25%.
 16. The method according toclaim 1, wherein the transfer is carried out at relative humidity ofabout 40% to 100%.
 17. The method according to claim 1, wherein thecomplementary nucleic acid is part of a probe.
 18. The method accordingto claim 1, wherein the complementary nucleic acid is part of a linkingstrand, wherein the linking strand links the nucleic acid on thesubstrate to a probe.
 19. The method according to claim 1, wherein thepattern of nucleic acid has a lateral dimension of about 500 nm or less.20. The method according to claim 1, wherein the pattern of nucleic acidhas a lateral dimension of about 200 nm or less.
 21. The methodaccording to claim 1, wherein the pattern of nucleic acid has a lateraldimension of about 10 nm to about 100 nm.
 22. The method according toclaim 1, wherein the pattern of nucleic acid has height of about 100 nmor less.
 23. The method according to claim 1, wherein the pattern ofnucleic acid is a dot.
 24. The method according to claim 1, wherein thepattern of nucleic acid is a line.
 25. The method according to claim 1,wherein after transfer the substrate is passivated in non-patternedareas.
 26. The method according to claim 1, wherein the nanoscopic tipis an atomic microscope tip, the nucleic acid is an oligonucleotidewhich comprises functional groups to provide for chemisorption orcovalent bonding to the substrate, and wherein the nucleic acid patternis a dot or line having, respectively, a dot diameter or a line width ofabout 500 nm or less.
 27. The method according to claim 1, wherein thenanoscopic tip is an atomic microscope tip, the nucleic acid is anoligonucleotide which comprises functional groups to provide forchemisorption or covalent bonding to the substrate, the nucleic acidpattern is a dot or line having, respectively, a dot diameter or a linewidth of about 500 nm or less, and, the transfer is carried out atrelative humidity of about 25% to about 50%.
 28. The method according toclaim 1, wherein the nanoscopic tip is an atomic microscope tip, thenucleic acid is an oligonucleotide which comprises functional groups toprovide for chemisorption or covalent bonding to the substrate, thenucleic acid pattern is a dot or line having, respectively, a dotdiameter or a line width of about 500 nm or less, the application iscarried out at relative humidity of at least about 25%, and the tip ismodified to have a positively charged surface.
 29. The method accordingto claim 1, wherein the nanoscopic tip is an atomic microscope tip or ahollow tip, the nucleic acid is a deoxyribose single stranded nucleicacid, the nucleic acid pattern is a dot or line having, respectively, adot diameter or a line width of about 10 nm to about 100 nm, and whereinthe substrate is also passivated after transfer in the non-patternedarea. 30.-129. (canceled)
 130. A nanoscale nucleic acid pattern on asubstrate comprising the substrate and at least one pattern of a firstnucleic acid on the substrate, wherein the pattern of first nucleic acidis chemisorbed or covalently bonded to the substrate, has a lateraldimension of 1,000 nm or less, and is hybridizable to a second nucleicacid complementary to the first, wherein the pattern of first nucleicacid is formed by the method according to claim
 1. 131. A nanoscalenucleic acid pattern according to claim 130, wherein the lateraldimension is 500 nm or less.
 132. A nanoscale nucleic acid patternaccording to claim 130, wherein the lateral dimension is 200 nm or less.133. A nanoscale nucleic acid pattern according to claim 130, whereinthe lateral dimension is 100 nm or less.
 134. A nanoscale nucleic acidpattern according to claim 130, wherein the pattern is a dot.
 135. Ananoscale nucleic acid pattern according to claim 130, wherein thepattern is a line.
 136. A nanoscale nucleic acid pattern according toclaim 130, wherein the pattern is hybridized to a probe.
 137. Ananoscale nucleic acid pattern according to claim 136, wherein the probeis a nanoparticle functionalized with a nucleic acid.
 138. A nanoscalenucleic acid pattern according to claim 130, wherein the pattern ishybridized to a linking strand comprising the second nucleic acid,wherein the linking strand is also hybridized to a probe.
 139. Ananoscale nucleic acid pattern according to claim 138, wherein the probeis a nanoparticle functionalized with a nucleic acid.
 140. A nanoscalenucleic acid pattern according to claim 130, wherein the first nucleicacid is chemisorbed to the substrate.
 141. A nanoscale nucleic acidpattern according to claim 130, wherein the first nucleic acid iscovalently bonded to the substrate.
 142. A nanoscale nucleic acidpattern according to claim 130, wherein the first nucleic acid is singlestranded DNA.
 143. A nanoscale nucleic acid pattern according to claim130, wherein the first nucleic acid is an oligonucleotide.
 144. Ananoscale nucleic acid pattern according to claim 130, wherein the firstnucleic acid comprises a spacer group, linking a functional group forchemisorption or covalent bonding to the substrate and the first nucleicacid.
 145. A nanoscale nucleic acid pattern according to claim 130,wherein the first nucleic acid is functionalized with an electrophilicgroup covalently bonded to the substrate.
 146. A nanoscale nucleic acidpattern according to claim 130, wherein the first nucleic acid isfunctionalized with an electrophilic group covalently bonded to thesubstrate, the substrate comprising a surface self assembled monolayerexposing nucleophilic groups for covalent bonding to the first nucleicacid.
 147. A nanoscale nucleic acid pattern according to claim 130,wherein the substrate is an electrical insulator.
 148. A nanoscalenucleic acid pattern according to claim 130, wherein the substrate is anelectrical insulator, wherein the first nucleic acid is functionalizedwith an electrophilic group covalently bonded to the substrate, thesubstrate comprising a surface self assembled monolayer exposingnucleophilic groups for covalent bonding to the first nucleic acid. 149.A nanoscale nucleic acid pattern according to claim 130, wherein thefirst nucleic acid comprises a spacer group, linking a functional groupfor chemisorption or covalent bonding to the substrate and the firstnucleic acid, wherein the first nucleic acid is an oligonucleotide, andwherein the lateral dimension is 200 nm or less. 150.-155. (canceled)156. A nucleic acid nanoarray comprising a substrate and a plurality ofpatterns of nucleic acid on the substrate, wherein the patterns ofnucleic acid are chemisorbed or covalently bonded to the substrate, havelateral dimensions of about 1,000 nm or less and are separated from eachother by distances of 1,000 nm or less, and have a pattern density of atleast 100,000 per square centimeter, and are hybridizable tocomplementary nucleic acids, and wherein the patterns of nucleic acidare formed by the method according to claim
 1. 157. (canceled)